Commercialization of a process making effective use of somatic embryogenesis is considered desirable over and economically more attractive than, for example, organogenic cloning because of the potential for higher yields of plants over comparatively short time intervals.
It is known that certain plant cells have the potential to differentiate into whole plants when cultured in appropriate plant tissue culture media. Such media typically comprise inorganic salts, a carbon source such as sucrose, inositol, thiamine, and the like. Examples of plant tissue culture media commonly used are those of Murashige and Skoog (MS medium), Lindsmaier and Skoog, Gamborg (B5 medium), and the like.
The composition of such plant tissue culture media may be modified to optimize the growth of the particular plant cells employed. Almost all plant cells require plant hormones e.g. auxins or auxin-like compounds such as indole acetic acid, indole butyric acid, naphthalene acetic acid, or 2,4-D, and/or a cytokinin such as benzyl adenine, zeatin, kinetin, or the like. In order to secure optimal growth it may also be advantageous to add vitamins such as nicotinic acid, pyridoxine or other components such as coconut milk, caseine hydrolyzates and the like.
In the past, the formation of true somatic embryos has required the use of callus material which comprises undifferentiated conglomerates of cells having very large vacuoles and smaller, round cells having very small vacuoles, as the primary growth phase material from which somatic embryos can then be derived. However, the use of such material as starting material in somatic embryogenesis has many drawbacks and has proven to be of limited use in the obtention of large numbers of plants having substantially similar phenotype and/or being substantially uniform with respect to ploidy level. Plants originating from callus have a tendency to display somaclonal variation and/or non-uniformity with respect to ploidy level [(Chaleff R. S. (1983) Science 219:676-682; Larkin P. J. (1987) Iowa State Journal of Research 61(4):393-434; De Klerk G-J (1990) Acta Bot. Neerl.39(2):129-144; Karp A. & Bright S. W. J. (1985) Oxford Surveys of Plant Molecular & Cell Biology 2:199-234; Custers J. B. M. et al (1990) Acta Bot. Neerl. 39(2):153-161 (cucumis sativus L.); Kysely W. et al (1987) Plant cell Reports 6:305-308 (pisum sativum L.); Ezura H. (1992) Plant Science 85:209-213 (cucumis melo); and Kiviharju E. et al (1992) Plant Cell, Tissue, and Organ Culture 28:187-194 (Cyclamen persicum Mill)].
Known examples of patent applications make use of callus material in somatic embryogenesis. An example, WO90/01058 to Plant Genetics Inc. describes using callus material to acquire somatic embryos while investigating the effect of employing a wide range of synthetic auxins. Callus material is formed or grown from suitable explant material over many weeks of culturing and/or subculturing on solid media. Somatic embryogenesis is then initiated by transferring callus tissue to a medium containing a plant hormone such as 2,4-D or an analogue thereof. No mention is made of the ploidy level of somatic embryos, or of the ploidy level of plants obtained. While the use of callus material in somatic embryogenesis may be helpful in obtaining plants in which the-obtention of somaclonal variants may be interesting for enriching an available gene pool, it is of little use for seed merchants or breeders who simply wish to obtain commercial numbers of plants which have substantially all the same genotype.
It is acknowledged that carrot cell lines have been cloned from microclusters composed of meristematic cells and studied for their capacity to produce embryos. (P. Coutos-Thevenot et al, Plant Cell Reports (1990) 8: 605-608).
The authors report employing an initial cell suspension from hypocotyls of domestic carrot (S1 strains) in a plant tissue culture medium comprising the auxin 2,4-D, isolating cell clusters by filtration, resuspending the cell clusters in plant tissue culture medium comprising 2,4-D to increase the cluster population density, transferring cell colonies from isolated clusters to a Petri dish comprising solid plant tissue culture medium containing 2,4-D and 1% bacto-agar to induce cell colony formation and depositing them in a 2nd solid medium containing 2,4-D and 1% bacto-agar around a nurse S1 strain callus, dissociating each cell colony in plant tissue culture medium containing 2,4-D and subculturing the thus obtained cultures in plant tissue culture medium containing 2,4-D. The subsequent analysis of the cell lines obtained according to this process revealed that 13 out of 40 cell lines were embryogenic, but most of these lines lost their embryogenic potential over time. Only one cell line had a rather constant embryogenic potential over a larger period. According to flow cytometric analysis the latter line was diploid.
The present invention provides a method of obtaining somatic embryos in suspension culture which is technically simple. It does i.a. not require the deposition of cell lines around a nurse callus. The method of the invention allows accordingly the production of somatic embryos at a commercial scale. The somatic embryos according to the invention, are capable of being used to provide commercial quantities of plants having substantially the same genotype.
Hitherto, somatic embryogenesis has been indicated as a potentially powerful tool in the obtention of plants, however, the impracticalities of utilizing somatic embryogenesis starting from callus material have prevented the successful exploitation of the technology.
It has now surprisingly been found that commercial quantities of true somatic embryos can be obtained through the employment of liquid culturing techniques per se and without the need to employ solid media and/or callus tissue. It has also been found that it is possible to increase the biomass of PEMs in liquid media and hence a capacity to produce somatic embryos in commercial quantities therefrom. Using such liquid culturing techniques obviates the need to employ callus culturing/callus sub-culturing steps and provides for the first time a means of obtaining populations of somatic embryos which are uniform with respect to ploidy level.
The present invention provides a method for obtaining somatic embryos via somatic embryogenesis which substantially reduces or eliminates the risk of obtaining somaclonal variants.
It provides non-Daucus somatic embryo suspension cultures wherein the ploidy level of somatic embryos contained therein is substantially uniform.
The invention further provides plants having a substantially uniform ploidy (e.g. diploid plants or tetraploid plants) derived from somatic embryo suspension cultures having substantially the desired ploidy level.
The invention provides a more reliable means of obtaining true somatic embryos in commercial quantities from explant material which does not rely on the employment of callus tissue and/or employ solid media as essential elements of the said means.
These and other objects of the invention will become apparent from a reading of the following description and examples.